Neutrophil elastase and cathepsin G are serine proteases stored within the primary granules of neutrophils. The activation and degranulation of neutrophils at inflammatory sites result in the release of these proteases where they encounter inhibitors that regulate their activity in certain inflammatory conditions. These proteases have been implicated in the pathogenesis of a variety of diseases. One example of an inflammatory disease associated with neutrophil recruitment is Chronic Obstructive Pulmonary Disease (COPD). COPD is a slowly progressive and incurable disease of the airways, characterized by a gradual loss of lung function. An estimated 20.9 million Americans are currently affected by COPD; of which 18.3 million suffer from chronic bronchitis and 2.7 million suffer from emphysema. Our Phase I study involved the development of two rationally designed mutants of Plasminogen Activator Inhibitor One (PAI-1). The normal targets for PAl-1 are the plasminogen activators tPA and uPA. The gene for PAl-1 has been altered to produce mutants with altered protease specificity. These two new mutant inhibitors now target neutrophil elastase and cathepsin G. Due to the unique properties of PAl-1, these mutants actually exhibit properties superior to the natural inhibitors Alpha One Proteinase Inhibitor and Antichymotrypsin. Specifically, these mutants both inactivate and facilitate the cellular endocytosis and degradation of the neutrophil proteinases in the presence of polyanionic surfaces such as heparin and DNA, which sequester these very basic enzymes. The DNA that codes for these mutants has been cloned into a E. coli bacterial construct for expression at very high levels, and a patent pending method for the purification of PAl-1 will be scaled up in our Phase II study. The Phase II proposal extends and expands our studies to include: improving the properties of the mutants for therapeutic use, further characterization of the performance of the mutants compared with commercially available and endogenous inhibitors in a number of animal models, development of sensitive and specific immunoassays for neutrophil elastase and cathepsin G and, a study to examine the large scale production of the PAl-1 mutants using a rapid patent pending purification process. Destructive lung diseases are a major cause of morbidity in the U.S. We believe that these mutants can be developed into therapeutic agents for the treatment of a variety of inflammatory diseases.
